Quantitative nature of the Prolamin-box, ACGT and AACA motifs in a rice glutelin gene promoter: minimal cis-element requirements for endosperm-specific gene expression

The -197 bp promoter of the rice seed storage protein gene, GluB-1, is capa ble of conferring endosperm-specific gene expression. This proximal 5' flan king region contains four motifs, GCN4, AACA, ACGT and Prolamin-box, which are conserved in many seed storage protein genes. We previously showed that multiple copies of GCN4 conferred endosperm expression pattern when fused to the -46 core promoter of CaMV 35S. In this paper we demonstrate, using a similar approach, that tandem repeated copies of any of the other three mo tifs are unable to direct expression in seeds as well as other tissues of t ransgenic rice plants. Mutational analysis of individual motifs in the -197 bp promoter resulted in remarkable reductions in promoter activity. These results indicate that the GCN4 motif acts as an essential element determini ng endosperm-specific expression and that the AACA, ACGT and Prolamin-box a re involved in quantitative regulation of the GluB-1 gene. A set of gain-of -function experiments using transgenic rice showed that either the Prolamin -box or AACA, although often coupled with GCN4 in many genes, is insufficie nt to form a functional promoter unit with GCN4, whereas a combination of G CN4, AACA and ACGT motifs was found sufficient to confer a detectable level of endosperm expression. Taken together, our results provide direct insigh t into the importance of combinatorial interplay between cis-elements in re gulating the expression of seed storage protein genes.